Thrombin and factor Xa are key enzymes of blood coagulation. Factor Xa (FXa) is activated from its precursor, factor X, by either the intrinsic tenase complex (factor IXa/factor VIIIa) or the extrinsic tenase complex (tissue factor/FVIIa). Factor Xa activates prothrombin to thrombin, a reaction that is enhanced 400,000-fold when FXa is incorporated into the prothrombinase complex consisting of factor Va, calcium and phospholipids. Thrombin catalyzes the conversion of fibrinogen to fibrin and activates platelets both of which results in the formation of blood clots. Thrombin has additional functions both within and outside the coagulation system. By activating factors VIII, V, and XI, thrombin amplifies its own generation whereas protein C activation by thrombin contributes to the downregulation of coagulation. Activation of factor XIII as well as the thrombin activatable fibrinolysis inhibitor (TAFI) by thrombin affect the fibrinolytic system and contribute to clot stabilization, and several cellular and inflammatory functions of thrombin are mediated mainly via binding to the protease-activated receptors.
Both thrombin and factor Xa are validated targets for anticoagulant therapies. The majority of anticoagulant drugs in clinical use have anti-thrombin or anti-FXa activity, or both. Direct thrombin inhibition attenuates fibrin formation, thrombin-mediated activation of factors V, VIII, XI and XIII, and thrombin-induced platelet activation and aggregation.
Factor Xa has become an attractive target for antithrombotic therapy because of its position upstream of thrombin in the sequence of coagulation reactions. The fact that activation of one factor Xa molecule results in the generation of 1000 molecules of thrombin suggests that small amounts of a factor Xa inhibitor can effectively block thrombin generation without the need for high systemic levels of antithrombotic drug concentrations while low levels of thrombin remain active to ensure primary hemostasis and other functions of thrombin. Selective factor Xa inhibition has been shown in numerous animal studies to provide antithrombotic efficacy with little or no effect on markers of primary hemostasis (Leadley et al., Curr. Top. Med. Chem. 1, 151-159, 2001).
Heparin targets multiple enzymes in the coagulation cascade including thrombin and factor Xa. It has been the mainstay of antithrombotic therapy for more than 60 years but its use is associated with a number of disadvantages. Limitations of heparin result from its indirect, antithrombin (AT)-dependent mode of inhibition as well as from non-specific binding to plasma proteins and cells. Low molecular heparins (anti-Xa and anti-thrombin activity) and the sulfated pentasaccharides (selective anti-Xa agents) lack do not display the same nonspecific binding affinities and have replaced unfractionated heparin in some clinical settings. However, it has been demonstrated that clot-associated thrombin and prothrombinase contribute to thrombus growth and thrombin generation (Orfeo et al., J. Biol. Chem. 283, 9776-9786, 2008 and Brufatto et al., J. Thromb. Haemost. 1, 1258-1263, 2003), but are protected against AT-dependent anticoagulants like heparin, LMWHs, and pentasaccharides (Weitz et al., J Clin. Invest. 86, 385-391, 1990).
Small molecule direct inhibitors that simultaneously target thrombin and factor Xa have the potential to attenuate thrombin generation and thrombin activity more effectively than AT-dependent anticoagulants. The concept of dual inhibition of thrombin and factor Xa is also supported by the findings of Gould et al. (J Thromb. Haemost. 4, 834-841; 2010) demonstrating a synergistic antithrombotic effect of combining low doses of a direct thrombin inhibitor and a direct factor Xa inhibitor in vitro and in an animal model of thrombosis. Since bleeding time was not increased compared to the additive effect of each drug alone, the authors suggest that direct inhibition of multiple coagulation enzymes may provide an improved efficacy-to-safety ratio. Results from a study comparing unfractionated heparin, LMWH, a pentasaccharide, and a direct selective factor Xa inhibitor in vitro further support the concept that polytherapeutic agents are more effective anticoagulants than certain single-target agents in preventing surface-induced clot formation (Montalescot and Walenga, Clin. Appl. Thromb. Hemost. 15, 183-196, 2009).
During the past 10 years an increasing number of small molecule, selective factor Xa and thrombin inhibitors has been published and summarized in several review articles.
Several synthetic inhibitors of the active site of factor Xa have been disclosed. Two classes of inhibitors are to be distinguished: oral inhibitors and inhibitors for parenteral use. Xarelto (Rivaroxaban) with a Ki (against FXa) of 0.4 nM, (Perzborn et al., J Thromb Haemost 3:514-21, 2005), launched in 2008, and Apixaban with a Ki (against FXa) of 0.08 nM (BMS 652247, claimed in WO-03026652; April 2003; Apixaban, an oral, direct and highly selective factor Xa inhibitor: In vitro, antithrombotic and antihemostatic studies, Wong et al., J. Thromb. Haemostasis, 6, 820-829, 2008), are examples of oral anticoagulants in clinical use or in clinical development.
Similarly, synthetic inhibitors of the active site of thrombin (factor IIa), so-called direct thrombin inhibitors (DTI) have been disclosed, such as Exanta (Ximelagatran; Eriksson et al., J. Thromb. Haemost. 1, 2490-2496, 2003) with a Ki 2 nM, which has been withdrawn from the market in 2006, and Pradaxa (Dabigatran) with a Ki of 0.41 nM; first claimed in WO-9837075; Baetz and Spinler, Pharmacotherapy, 28, 1354-1373, 2008).
Argatroban is a small molecule DTI based on arginine, with Ki of 27-39 nM (Berry et al., Br. J. Pharmacol. 113, 1209-14, 1994). Examples of parenteral DTI in development are Melagatran (discontinued; Ki 1.3 nM), Flovagatran (Paion), or NU172 (Nuvelo) (Gross and Weitz, Clin Pharmacol Therapeut 86, 139-146, 2009; Weitz. Thromb. Haemost. 103, 62-70, 2010).
Similarly, there are selective direct FXa inhibitors in different stages of development such as Otamixaban (Guertin et al. Bioorg. Med. Chem. Lett. 12, 1671-1674, 2002) and selective peptidomimetic FXa inhibitors (Donneke et al., Bioorg. Med. Chem. Lett. 17, 3322-3329, 2007).
Stürzebecher et al. have described a series of N-terminal sulfonylated benzamidine peptidomimetics having various effects on serine proteases. Included within this class are factor Xa inhibitors, useful as anticoagulants and antithrombotics (U.S. Pat. No. 6,841,701); urokinase inhibitors, useful as tumor suppressors (US Pat. Application Publication No. 2005/0176993, U.S. Pat. No. 6,624,169); inhibitors of plasma kallikrein (PK), factor XIa and factor XIIa, useful as anticoagulants and antithrombotics (US Pat. Application Publication No. 2006/0148901); and matriptase inhibitors, useful as tumor suppressors (US Pat. Application Publication No. 2007/0055065). Clinical use of these inhibitors has not been reported.
A common feature of all these DTI and FXa inhibitors is their pronounced specificity of inhibition towards only one enzyme, either thrombin or FXa. Whereas unfractionated heparin (UFH) inhibits thrombin and FXa to similar extents, numerous FXa inhibitors and in particular sulfated glycosaminoglycans based on a reduction in the chain length as compared to low molecular weight heparin (LMWH), such as the clinically used Arixtra (Fondaparinux), Fragmin (Dalteparin) or Danaparoid present greater selectivity towards the inhibition of FXa (Eikelboom and Weitz, Circulation, 121, 1523-1532, 2010). Idrabiotaparinux, which is a modified fondaparinux with an antidote recognition moiety, is also an example of an indirect FXa inhibitor. In contrast to LMWH, fondaparinux and the mono-selective thrombin or FXa inhibitors, UFH indirectly inhibits not only thrombin and FXa, but also factors XIa and, to a lesser extent, XIIa and is thus effective in modulating the contact activation pathway. “This might explain in part why early attempts to use LMWH to prevent clotting in cardiac bypass circuits did not show any promise and why the risk of thrombosis of cardiac catheters is higher with fondaparinux than with UFH.” (Hirsh et al., Circulation 116, 552-560, 2007).
In contrast to inhibitors with a pronounced mono specificity, the concept of a dual inhibitor bears attractive resemblance to natural inhibitors of coagulation, namely heparin which inhibits both thrombin and FXa and has equal activity against both enzymes. None of the drawbacks or adverse effects of heparin has been attributed to its multiple mode of inhibition. Moreover, its multi-target activity, also involving inhibition of contact phase proteases, might be an advantage in situations of blood contact with foreign surfaces without contributing to hemorrhagic effects. The ability of the newly developed monoselective synthetic FXa and thrombin inhibitors to reproduce the favorable effects and therapeutic profile of heparin has been questioned (Fareed et al., Semin. Thromb. Hemost. 34, 58-73, 2008). In major randomized trials, selective agents so far have not demonstrated superiority over UFH or LMWH with regard to ischemic endpoints suggesting that compounds with multiple sites of action, like UFH or LMWH, result in better ischemic outcome in patients with acute coronary syndrome (ACS) (Cohen, Am. J Med. 123, 103-110, 2010). Selective agents like the tissue factor/factor VIIa inhibitor rNAPc2 and the selective indirect FXa inhibitor fondaparinux, have shown insufficient antithrombotic activity in ACS patients undergoing PCI (Chan et al., J Thromb. Thrombolysis 28, 366-380, 2009). Increased incidence of catheter thrombosis with fondaparinux compared to UFH suggests that additional thrombin inhibition is required to prevent contact-mediated activation, a phenomenon that is even more relevant in cardiopulmonary bypass (CPB) surgery.
Data from a study comparing UFH, LMWH, fondaparinux and otamixaban in vitro support the concept that polytherapeutic agents, including UFH and enoxaparin, are more effective anticoagulants than certain single-target agents in preventing surface-induced clot formation (Montalescot and Walenga, Clin. Appl. Thromb. Hemost. 15, 183-196, 2009). Dual inhibition of thrombin and FXa has the potential to effectively suppress thrombin generation and thrombin activity. A synergistic antithrombotic effect by simultaneous inhibition of thrombin and FXa has also been demonstrated in in vitro and animal models, and a ratio of anti-Xa/anti-thrombin activity of 2-3 has been found to be optimal with regard to efficacy and bleeding (Gould et al., J. Thromb. Haemost. 4, 834-841, 2006).
Tanogitran (Linz et al., WO 2004/000818; Ries et al., WO 2004/000310), which is characterized by an FXa/thrombin ratio of 0.1, RWJ445167 (Tianbao et al., U.S. Pat. No. 7,402,586; Maryanoff et al., Chem. Biol. Drug Des. 68:29-36, 2006)) with a ratio of <0.02, a series of oxazolopyridine based dual inhibitors (Deng et al., Bioorg. Med. Chem. Lett. 15, 4411-4416, 2005), and a series of quinoxalinone based dual inhibitors (Ries et al., Bioorg. Med. Chem. Lett. 13, 2297-2302, 2003) are dual thrombin/FXa inhibitors disclosed in the literature. There are also two products based on LMWH: M118 (Momenta; Kishimoto et al., Thromb. Haemost. 102, 900-906, 2009) and EP217609, (Endotis Pharma; Petitou, et al., Thromb. Haemost. 102, 804-810, 2009), both of which are equipotent against thrombin and FXa and share the specific feature that their action can be controlled by an antidote.